U.S. patent number 9,580,373 [Application Number 14/646,198] was granted by the patent office on 2017-02-28 for extraction and purification of urushiol from botanical sources.
This patent grant is currently assigned to Rowan University. The grantee listed for this patent is Rowan University. Invention is credited to Ryan Elliot, Nikita Iltchenko, Weixing Li, Catherine F. Yang.
United States Patent |
9,580,373 |
Yang , et al. |
February 28, 2017 |
Extraction and purification of urushiol from botanical sources
Abstract
The disclosure relates to methods for preparing urushiol from
plants including, for example, poison ivy or poison oak. The
methods include extraction of plant material using a primary
organic solvent more polar than ethanol, followed by a solvent
extraction using substantially immiscible solvents having
substantially different polarities, such as hexane and
acetonitrile. The method can include further purification, such as
by fractionation of solvent-extracted materials using a
thiazole-derivatized silica gel chromatography medium. The extracts
thus generated can exhibit greater purity, higher concentration,
and greater stability than extracts made using previously-known
methods. The extracts can be particularly suitable for use in
immunotherapeutic methods, such as desensitizing individuals who
normally develop allergic contact dermatitis attributable to poison
ivy or poison oak.
Inventors: |
Yang; Catherine F. (Glassboro,
NJ), Li; Weixing (Glassboro, NJ), Elliot; Ryan
(Glassboro, NJ), Iltchenko; Nikita (Glassboro, NJ) |
Applicant: |
Name |
City |
State |
Country |
Type |
Rowan University |
Glassboro |
NJ |
US |
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Assignee: |
Rowan University (Glassboro,
NJ)
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Family
ID: |
50776573 |
Appl.
No.: |
14/646,198 |
Filed: |
November 21, 2013 |
PCT
Filed: |
November 21, 2013 |
PCT No.: |
PCT/US2013/071355 |
371(c)(1),(2),(4) Date: |
May 20, 2015 |
PCT
Pub. No.: |
WO2014/081988 |
PCT
Pub. Date: |
May 30, 2014 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20150284308 A1 |
Oct 8, 2015 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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61729081 |
Nov 21, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01D
11/02 (20130101); B01D 11/0288 (20130101); C07C
35/14 (20130101); A61K 36/22 (20130101); A61K
2236/00 (20130101) |
Current International
Class: |
C07C
35/14 (20060101); B01D 11/02 (20060101); A61K
36/22 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Gartner, Barbara L., et al., Seasonal Variation of Urushiol Content
in Poison Oak Leaves, "American Journal of Contact Dermatitis",
Mar. 1993, vol. 4, No. 1, pp. 33-36. cited by applicant .
Ki Tae Suk et al., In vitro Antibacterial and Morphological Effects
of the Urushiol Component of the Sap of the Korean lacquer tree
(Rhus vernicifera Stokes) on Helicobacter pylori J. Korean Med Sci.
2010; pp. 399-404. cited by applicant.
|
Primary Examiner: Davis; Brian J
Attorney, Agent or Firm: Fox Rothschild LLP
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
This application is the U.S. National Phase filing of PCT
International Application No. PCT/US13/71355, filed Nov. 21, 2013,
which claims priority to U.S. Provisional Application No.
61/729,081, filed Nov. 21, 2012. The contents of the foregoing
applications are incorporated herein by reference in their
entireties.
Claims
What is claimed is:
1. A method for extracting urushiol from a plant, the method
comprising contacting the plant and a primary organic solvent that
is more polar than ethanol for a period of time and thereafter
separating the plant from the primary solvent to yield a liquid
initial extract; removing the primary solvent from the initial
extract to yield a crude extract; extracting the crude extract with
a mixture of first and second solvents, the first and second
solvents being immiscible and the first solvent being more polar
than the second solvent; and recovering urushiol from the first
solvent.
2. The method of claim 1, wherein the primary organic solvent is
selected from the group consisting of methylene chloride,
isopropanol, tetrahydrofuran, dichloromethane, acetonitrile,
dimethoxyethane, propanol, chloroform, and pentane and combinations
thereof.
3. The method of claim 1, wherein the primary organic solvent is
selected from the group consisting of methylene chloride,
chloroform, and pentane.
4. The method of claim 1, wherein the first solvent is no more
polar than chloroform.
5. The method of claim 1, wherein the first solvent is selected
from the group consisting of acetonitrile and chloroform.
6. The method of claim 1, wherein the second solvent is more polar
than chloroform.
7. The method of claim 1, wherein the second solvent is selected
from the group consisting of alkanes having at least five carbon
atoms.
8. The method of claim 1, wherein the second solvent is selected
from the group consisting of pentanes, hexanes, and heptanes.
9. The method of claim 1, wherein the plant is selected from the
group consisting of cashews, pistachios, mangos, poison ivies,
poison oaks, sumacs, smoke trees, marulas, yellow mombins,
cuachalalates, lac trees, rengus trees, Burmese lacquer trees,
India marking nut trees, ginkgo trees, and combination of
these.
10. The method of claim 1, wherein the plant is one of poison ivy
and poison oak.
11. The method of claim 1, wherein the primary organic solvent is
contacted with leaves of the plant.
12. The method of claim 1, wherein the plant is shredded.
13. The method of claim 1, wherein each step is performed at a
temperature not greater than 30 degrees Celsius.
14. The method according to claim 1, further comprising
fractionating urushiol recovered from the first solvent using a
thiazole-derivatized chromatography medium.
15. The method of claim 14, wherein the medium is a silica gel.
Description
BACKGROUND OF THE DISCLOSURE
The disclosure relates generally to the field of immunology and
allergic reactions.
Poison ivy is one of the most medically problematic plants, and is
responsible for causing allergic contact dermatitis in humans. It
has been estimated that approximately 85% of the US population is
sensitized to these plants. While the chemistry of the allergen and
the immunologic mechanism of the reaction are different, both
clinical experience and animal studies suggest that poison ivy,
like respiratory allergy to ragweed, should be responsive to
injection immunotherapy. Ragweed allergen was identified and
quantitated in the 1960's and its safe and effective immunotherapy
doses identified in dose ranging studies in the 1960`s and`70's.
Immunotherapy has the prospective ability to lessen the abnormal
immunological responses to an allergen. Double blind immunotherapy
discontinuation studies of ragweed have shown that even after a
full year patients show no symptoms of response to the plant. There
are no known studies to validate human injection immunotherapy or
identify safe and effective treatment doses and schedules for
poison ivy allergies.
Urushiol is an oily organic allergen found in plants in the family
Anacardiaceae. Notable plants includes cashew (in the type genus
Anacardium), mango, pistachio, poison ivy (e.g., Toxicodendron
radicans), poison oak (e.g., Toxicodendron diversilobum and
Toxicodendron pubescens), sumac, smoke tree, marula, yellow mombin,
and cuachalalate. It has been demonstrated that the heteroolefinic
components of the urushiols contained in the plants within this
family are not identical in structure or composition. The term
urushiol as used herein includes mixtures of heteroolefinic
catechols of Anacardiaceae plants, e. g., poison ivy, poison oak,
cashew, pistachio, or mango, as well as to individual components of
such mixtures.
Urushiol is a mixture 3-n-alk-(en)-yl catechols with varying
degrees of unsaturation on either the C.sub.15 side chain (derived
from poison ivy chain) or C.sub.17 side chain (derived from poison
oak), often including those shown in FIG. 1. Conclusive
identification of the structure was first made in 1934;
purification yielded a yellow oil with a boiling point of 210
degrees Celsius. Urushiol primarily resides in resin canals,
present in the leaves, flowers, roots, bark and fruits of the
plant. In addition to poison oak, poison ivy, and poison sumac,
other urushiol-containing plants in this family are the Chinese and
Japanese lac trees (R. verniciflua L.) and the Yunnan, Formosan and
Indo-Chinese lac trees (R. succedanea L.). Agriculturally
significant Mango (M. indica), Cashew (A. occidentale), and
Pistachio (P. vera) trees also contain urushiol.
The long aliphatic side chains of these catechols cause the
molecules to be hydrophobic, making urushiol one of the most
lipophilic haptens that cause contact dermatitis. As a result of
this lipophilicity urushiol tends to concentrate in cellular
membranes. Upon contact with human tissue, the catechol ring
undergoes oxidation, forming an electrophilic o-quinone. This
undergoes a regiospecific attack by model sulfhydryl and amino
nucleophiles present on proteins to form the adduct. However, the
lack of covalent bonding between urushiol and cell membranes used
for introduction into cultures makes the mechanism of antigen
presentation unclear.
In vivo experiments suggest that T-cell receptors may be directed
against the side chain, with the di-saturated and tri-saturated
chains producing the majority of the immunological response. Yet
due to the hydrophobic nature of the molecule, the alkyl side chain
should be buried within the membrane leaving the catechol ring near
the surface. Therefore it is difficult to theorize how the side
chain could move out of the membrane and have an effect upon
immunological response. One proposed mechanism attributes it to a
membrane vesicle. Alternatively T-cell reaction could be against
the catechol ring, and increasing reactivity with unsaturated side
chains could be due to an altered presentation of the catechol
ring.
Immunotherapy with water-soluble ragweed antigen E has been shown
to not only prevent reactions on natural exposure during
immunotherapy but also to provide durable protection for at least a
year after stopping treatment. Double blind immunotherapy
discontinuation studies of ragweed have shown that, even after a
full year, patients show no symptoms of response to the antigen.
Based on immunological studies of ragweed, it was hypothesized that
urushiol can be extracted and used in immunotherapy against contact
dermatitis. However, previous extraction methods have proven
difficult and inefficient, being multi-stepped and involving
multiple solvents. The most common technique used by others
involves soaking plant material in ethanol followed by distillation
or dry packed silica column chromatography. Urushiol congeners can
be separated using HPLC if desired. An example of an urushiol
extraction method (hereinafter the "ElSohly method") described by
ElSohly et al. (J. Nat. Prod., 1982, 45(5):532-538) in summarized
in FIG. 2.
The ElSohly method and other multiple-solvent purification methods
have shortfalls, including use of undesirably large quantities of
solvents, restriction to small (volumetric) scale processing, and
low yields. Limitations of crude ethanol extracts as allergy
vaccines include limited storage stability and urushiol
concentrations insufficient for effective treatment of moderately
sensitive individuals.
Crude ethanol extracts having urushiol concentrations up to 1-2
milligram per milliliter have been described. (Coifman R E,
"Successful immunotherapy for poison ivy," presented to 2010 Annual
Meeting of the American Academy of Allergy Asthma & Immunology,
New Orleans La., 28 Feb. 2010 (Abs. 142), epub J. Allerg. Clin.
Immunol. 2010 (February)).
A need exists for improved methods for extraction of urushiol from
plant materials at concentrations and purity levels suitable for
immunotherapy or other treatment of hypersensitivity.
SUMMARY OF THE DISCLOSURE
This disclosure relates to a method for preparing a concentrated
urushiol extraction of a plant, comprising soaking materials from
the plant in at least one primary organic solvent selected from the
group consisting of methylene chloride, isopropanol,
tetrahydrofuran (THF), dichloromethane (DCM), acetonitrile,
dimethoxyethane, propanol, chloroform, and pentane, or combinations
of these, filtering out the plant materials forming an initial
extract, removing the primary solvent from the initial extract
forming a crude extract, combing the crude extract with hexane
forming a resuspension, extracting the resuspension with
acetonitrile forming an acetonitrile fraction and a depleted
fraction, and removing acetonitrile from the acetonitrile fraction
producing a concentrated urushiol extract.
The plant can be selected from the group consisting of cashews,
pistachios, mangos, poison ivies, poison oaks, sumacs, smoke trees,
marulas, yellow mombins, cuachalalates, lac trees, rengus tree,
Burmese lacquer tree, India marking nut tree, ginkgo biloba, and
combination of these. The materials from the plant may be leaves,
stems, bark, or shells.
The materials from the plant can be shredded before soaking in the
primary solvent. The soaking of materials from the plant may also
including actively mixing (e.g., stirring or shaking) the materials
and the primary solvent.
The extraction method or portions of it are preferably performed at
or below 30 degree Celsius.
The disclosure herein also relates to a method for preparing a
concentrated urushiol extraction of a plant. That method includes
fractioning an urushiol preparation using a thiazole-derivatized
chromatography medium, such as a thiazole-derivatized silica gel (a
"thiazole silica gel"). Fractionating with thiazole silica gel
involves contacting an urushiol preparation with thiazole silica
gel, and eluting urushiol therefrom using chloroform (e.g., by
applying to the gel a fluid having an increasing proportion of
chloroform over time). The fractionating with thiazole silica gel
may be performed at a sub-atmospheric pressure, such as under
ordinary laboratory "vacuum" conditions.
The disclosure also relates to a highly purified urushiol extract
of a plant having a purity of more than 96% (w/w). The highly pure
urushiol extract may be produced and purified by the aforementioned
methods.
BRIEF SUMMARY OF THE SEVERAL VIEWS OF THE DRAWINGS
FIG. 1 is a series of chemical structures of urushiol congeners
that occur in poison ivy and poison oak. Individual congeners that
are shown include, for poison ivy: 1a is pentadecyl catechol (PDC),
1b is monounsaturated (8-9) PDC, 1c is diunsaturated (8-9, 11-12)
PDC, and 1d is triunsubstituted (8-9, 11-12, 14-15) PDC: and for
poison oak: 2a is heptadecyl catechol (HDC), 2b is monounsaturated
(8-9) HDC, 2c is diunsaturated (8-9, 11-12) HDC, and 2d is
triunsubstituted (8-9, 11-12, 14-15) HDC.
FIG. 2 illustrates a previously described method of preparing
urushiol.
FIG. 3 illustrates a method described herein for preparing
urushiol, using methylene chloride as the primary solvent.
FIG. 4 is a gas chromatograph profile of a poison ivy urushiol
extract prepared as described herein.
FIG. 5 is a graph that illustrates stability over time of a poison
ivy urushiol extract prepared as described herein.
FIG. 6 is a depiction of % urishiol, wherein Diamonds represent
samples stored at 3 degrees Celsius, and Rectangles represent
samples stored at 23 degrees Celsius.
DETAILED DESCRIPTION
The disclosure relates to methods of preparing urushiol and related
compounds from botanical sources such as plants of the
Anacardiaceae family, including poison ivy and poison oak. In
particular, this disclosure relates to extraction of urushiol to
produce a substantially purified urushiol product, including
products suitable to diagnose and treat allergic contact
dermatitis, such as that resulting from contact with poison ivy or
poison oak, or suitable for use as a vaccine or immunotherapy
agent.
Within the context of this disclosure, the term "vaccine" or
"allergy vaccine" refers to a compound that can induce tolerance to
undesirable cell-mediated immunity conditions, such as allergic
contact dermatitis. The methods described herein can be adapted for
extraction of urushiol from plants of the family Anacardiaceae and
from other plants, such as cashew, pistachio, mango, Rengus tree,
Burmese lacquer tree, India marking nut tree, and the shell of the
cashew nut, as well as ginkgo biloba.
Three urushiol catechols have an unsaturated side chain and these
catechols are believed to be progressively less stable than the
saturated fourth component, 3-n-pentadecyl catechol, and thus more
prone to polymerization and oxidation reactions as the degree of
unsaturation in the side chain increases from one to three olefinic
bonds. Aged samples of urushiol have variable compositions of these
unsaturated components along with their polymers and oxidation
products, depending upon the degree of prior exposure to air,
water, and light, as well as the presence of acidic or basic
conditions and elevated temperatures.
Unsaturated components of urushiol show a tendency to polymerize
under acidic conditions and to oxidize under aerobic conditions,
with oxidation being accelerated in an alkaline environment. These
reactions lead to products of a polymeric nature and with a loss in
aromaticity. For these reasons, elevated temperatures and exposure
to air should be avoided during the extraction process. It is also
advisable to use freshly collected raw materials when extracting
urushiol from plant sources.
The methods of preparing urushiol described herein include
extraction with a primary organic solvent to form an initial
extract, stripping the primary solvent from the initial extract to
yield a crude extract and prior to solvent extraction using a
mixture of a more-polar first solvent and a less-polar second
solvent that are substantially immiscible with one another under
the extraction conditions used (e.g., hexane and acetonitrile used
at a temperature<30 degrees Celsius).
The primary solvent should be less polar, and preferably more
hydrophobic, than ethanol. However, a few relatively hydrophilic
solvents (e.g., acetonitrile and tetrahydrofuran) can yield
acceptable results Examples of suitably non-polar and hydrophobic
solvents include methylene chloride, isopropanol, dichloromethane,
dimethoxyethane, propanol, chloroform, pentane, and mixtures of
these.
An example of the preparative method using methylene chloride as
the primary solvent is shown in FIG. 3. In that method, the primary
solvent used in the initial extraction is removed by evaporation to
yield the crude extract. The crude extract is resuspended in hexane
(or another aliphatic hydrocarbon solvent) and acetonitrile.
Because those two solvents are immiscible under the conditions
used, materials in the crude extract can partition between the
immiscible solvent phases. The urushiol extract from the
acetonitrile fraction can then be concentrated, and acetonitrile
removed by evaporation, forming a concentrated urushiol extract.
The concentrated urushiol extract can then be resuspended in a
suitable solvent, such as ethyl alcohol, and stored. Concentrated
urushiol can be separated into congeners by means of high pressure
liquid chromatography or other similar methods, if desired.
In the methods used in the examples described herein, processing
steps were performed at room temperature (i.e., ca. 20 degrees
Celsius). The preparative procedures are preferably performed at
temperatures below 30 degree Celsius.
The ElSohly method includes multiple steps, including ethanol
extraction of poison ivy leaves, followed by chloroform/water
fractionation, dry-packed silica-gel column chromatography, and
hexane/acetonitrile partitioning. The extraction methods disclosed
herein have the advantage of utilizing a single primary solvent and
fewer overall steps. Use of single solvents can avoid toxicity in
humans. The methods described herein also can achieve higher yield
and purity with fewer purification steps than prior methods, and
improve the stability of the extraction products. The methods
described herein typically provide yields of urushiol in the range
of 1.33-1.43 grams per kilogram of raw material (i.e., a
0.133-0.143% (w/w) yield).
The purity of the urushiol prepared according to the method
disclosed herein was higher, as determined by gas
chromatography-mass spectrometric (GCMS) analysis, than the
reported purity of urushiol extract using the ElSohly method. The
high purity urushiol extract obtained by the methods disclosed
herein is indicated by the gas chromatography profile shown in FIG.
4. Furthermore, the concentration of urushiol prepared according to
the methods disclosed herein is higher than previously achievable.
The ElSohly method has been demonstrated to yield an urushiol
extract having a concentration not greater than about 2 milligram
per milliliter. The concentration of urushiol in extracts made
using the process described herein was shown to be upwards of 50
milligram per milliliter.
These improved characteristics are highly relevant to important
intended uses of urushiol preparations, such as reagents for
immunologic treatment of humans who experience allergic reactions
upon contact with poison ivy or poison oak. Effective treatment
depends heavily upon the sensitivity of the individual seeking
treatment and the concentration and purity of urushiol
concentrations that are available for treatment. As the
individual's sensitivity to urushiol decreases, more urushiol is
needed to elicit a desired degree of reaction and a higher dose of
urushiol extract is needed to achieve successful immunotherapy
treatment. A higher dose must come from a higher concentration of
urushiol in the extract, and not from simply increasing the volume
of the urushiol extract, because there are physiological limits to
the quantity of reagent that can be effectively delivered to a
patient. It is therefore preferable to use immunotherapy agents
having high urushiol concentration and low solvent content.
The high concentration urushiol that can be prepared using the
methods described herein facilitate immunological treatment of
people who exhibit moderate or low sensitivity to poison ivy or
poison oak. The urushiol preparation obtained using the methods
described herein permits a much higher amount of urushiol to be
practically applied in order to elicit an allergic response in
these patients.
The urushiol extract prepared as described herein can be further
purified by chromatography, if desired. A preferred chromatographic
method is liquid chromatography employing a bed packed with a
thiazole-derivatized medium, such as the thiazole silica gel
described in the examples herein. Thiazole silica gel can be
prepared by reacting a silica gel by reacting with a thiazole
compound, for example, 5-(4-(trimethoxysilyl)butyl)thiazole. The
thiazole silica gel may be dry-packed in a column using known
methods. An urushiol sample can be applied to the dry-packed
thiazole silica column and eluted therefrom by passing over the
column packing a solvent that includes a solvent (e.g., chloroform)
that will displace urushiol from the thiazole-derivatized medium.
The chloroform or other solvent can thereafter be removed by
evaporation. If necessary or desired, trace amount of chloroform or
other solvent can be removed by ether distillation.
Compared to the unmodified silica gel used in the ElSohly method,
thiazole-modified silica gel enhances purification efficiency.
Urushiol extract can be purified using thiazole silica gel
chromatography to a purity greater than 96% by weight (ignoring
solvent) or higher, for example, above 97%, above 98%, or above
99%.
Table 1 summarizes examples of urushiol preparations from poison
ivy leaves that were performed as described herein. Examples 2-10
illustrate the methods described in this disclosure and generally
exhibited higher yield and greater urushiol concentration than the
ElSohly method.
Comparison of gas chromatography peak distributions and relative
abundances the effect of varying extraction routes on relative
congener/isomer concentrations, as shown in Table 2. Relative
congener concentration can also vary with the identity and
treatment of plant raw materials used in the extraction (for
example, the plant age, the season or time of year collected, and
the plants' specific growth conditions). In all performed
extraction routes the monounsaturated urushiol was in highest
concentration (.about.43%), averages of percentage concentration
varying statistically insignificantly among different extractions
but having a relatively high standard deviation within each data
set (as high as .+-.11% for pentane extractions) (Table 2). The
next most prominent congener (11-25%) was the di-unsaturated
urushiol which varied among the different extractions (Table 2).
The fully saturated congener, was present in less concentration
(5-6%) without variation among reactions (Table 2). The final
urushiol congener, the thrice disaturated molecule was present in
nearly undetectable levels throughout all extracts (1-3%) (Table
2). Ethoxy derivatives of urushiol as well as E/Z isomers varied
significantly among extractions suggesting their levels are greatly
dependent on the technique and care used during the purification,
as well as age and other factors.
TABLE-US-00001 TABLE 1 Summary of Examples of Poison Ivy Urushiol
Extraction and Purification Total Poison Ivy Leaf Crude Purity
Acetonitrile Weight Final Purity Example Primary Solvent Steps
Weight (g) (wt %) Fraction Purity (wt %) (g) (wt %) 1 Ethanol 5 30
42 ~42 0.25 90 2 Methylene Chloride 3 90 58 58 0.62 99.5 3
Isopropanol/THF 3 30 32 92 0.35 98 4 DCM/THF 3 29 30 90 0.30 N/A 5
THF 3 35 35 93 0.36 97.5 6 Acetonitrile 3 32 34 93 0.31 96.5 7
Dimethoxyethane 3 60 34 93 0.62 98.5 8 Propanol 3 60 34 93 0.60
97.5 9 Chloroform 3 35 67 90 0.58 94.5 10 Pentane 3 40 42 86 0.39
91.5
TABLE-US-00002 TABLE 2 Peak analysis of gas chromatographs of
average purified methylene chloride, chloroform and pentane
extracts shown with standard deviation. Urushiol Pentane Chloroform
Methylene Peak Congener (%) (%) Chloride (%) 1 1d 1.5 .+-. 0.9 2.7
.+-. 0.6 2.7 .+-. 0.9 2 1c 43 .+-. 11 43 .+-. 8.7 44 .+-. 4.0 3 1b
15 .+-. 2.9 25 .+-. 5.4 12 .+-. 3.8 4 1a 6.1 .+-. 1.1 6.5 .+-. 1.4
5.3 .+-. 0.9 5 1a 1.9 1.5 .+-. 0.1 2.1 .+-. 0.1 6 1c 3.3 .+-. 2.2
1.9 .+-. 1.2 3.2 .+-. 4.0 7 2c 1.5 .+-. 0.4 2.2 .+-. 1.6 3.0 .+-.
1.0 8 2b 4.3 .+-. 3.2 5.5 .+-. 4.0 11 .+-. 6 9 2a 6.5 .+-. 1.8 3.1
.+-. 0.7 9.2 .+-. 5.4
Urushiol in preparations made as described herein have shown
remarkable stability. Urushiol is stable at a concentration of 50
milligram per milliliter in ethanol. FIG. 5 shows the stability of
an urushiol extract prepared using methylene chloride as the
primary solvent. Samples were tested up to 250 days stored at 3
degrees Celsius (Diamonds) and 23 degrees Celsius (Squares). No
significant difference was observed over the time period for either
set of stored samples.
Using high concentration urushiol preparations made as described
herein, vaccines can be prepared that are suitable to induce
tolerance in patients with significant clinical disease, but with
relatively low levels of pre-treatment patch-test sensitivity. Both
clinical tolerance to natural exposure and reduction in patch test
sensitivity have lasted nine months or more using such vaccines.
These vaccines are believed to be the first to induce long-lasting
tolerance to urushiol in previously sensitized humans.
The subject matter of this disclosure is now described with
reference to the following Examples. The Examples are provided for
the purpose of illustration only, and the subject matter is not
limited to these Examples, but rather encompasses all variations
which are evident as a result of the teaching provided herein.
The methods described herein are illustrated in the following
example. While the example involves the preparation of urushiol
from poison ivy, an essentially similar procedure is followed in
preparing urushiol from poison oak or from other urushiol-bearing
plants.
Plant Material
Poison ivy (Toxicodendron radicans) leaves were collected in June,
July and August 2011 in a forest located in Mantua, N.J. The leaves
were stored in a refrigerator substantially immediately after
harvest and used for extraction within 7-10 days. Freshly collected
poison ivy raw material (leaves and stems) was torn by hand only to
the extent that each leaf was torn at least once before solvent
extraction. It is expected that more thorough shredding of such
materials will enhance extraction yield.
EXAMPLE 1
Poison ivy leaves (30 grams) were soaked in 100% ethanol (200
milliliters) for approximately 4 hours at room temperature. The
resulting solution was filtered to remove the leaves, collected in
a round bottom flask, and stripped of solvent on a rotary
evaporator. GCMS analysis showed approximately 42% w/w urushiol.
The resulting dark green residue was then resuspended in 100
milliliters chloroform and washed thrice with water. The organic
fractions were dried over sodium sulfate (10 gram) to remove traces
of water. The solvent was removed on a rotary evaporator with
temperature between 20 and 30 degrees Celsius to yield a residue.
That residue was fractionated by column chromatography (15
centimeter length by 4 centimeter diameter) with dry packed silica
gel under laboratory vacuum and eluted with chloroform. Fractions
were collected based on color and tested for urushiol presence by a
ferric chloride test and GCMS.
Fractions that tested positive for catechol were combined, stripped
of solvent, and resuspended in 100 milliliters hexane. The
resuspended material was thrice extracted with 100 milliliter
aliquots of acetonitrile. The acetonitrile aliquots were combined
and evaporated, yielding 0.25 gram of dark green oil. GCMS analysis
showed 90% w/w urushiol. The concentrated extract was resuspended
in ethanol and stored in refrigerator (3 degrees Celsius) in order
to slow the degradation process.
EXAMPLE 2
Poison ivy leaves and stems (90 grams) were soaked in methylene
chloride (800 milliliters) for 4.5 hours at room temperature. The
solution was filtered and stripped of solvent. GCMS analysis showed
58% w/w urushiol. The resulting dark brown oil (0.997 g) was
resuspended in ethanol (100 milliliters) and dried over sodium
sulfate (10 gram). The solution was then split into 3 equal volumes
and each fraction was stripped of solvent. Dark brown oil (0.332 g)
was resuspended in 100 milliliters hexane and extracted with
acetonitrile (100 milliliters) in a Soxhlet continuous extraction
apparatus. Acetonitrile fractions were collected and the solvent
was evaporated to yield a green-brown oil (0.62 gram). GCMS
analysis, showed 93% w/w urushiol.
Further purification was achieved by column chromatography (15
centimeter length by 4 centimeter diameter) with dry packed
thiazole silica gel eluted with chloroform under laboratory vacuum.
The purity was 99.5%. The solution was resuspended in ethanol and
placed in sealed amber vials under refrigeration (3 degrees
Celsius) to be formulated for clinical use.
EXAMPLE 3
Poison ivy leaves (30 grams) were soaked in 1:1 isopropanol:THF
(200 milliliters) for approximately 4 hours at room temperature.
The resulting solution was filtered to remove the leaves, collected
in a round bottom flask, and stripped of solvent using a rotary
evaporator. GCMS analysis showed approximately 32% w/w urushiol.
The resulting dark green residue was resuspended in 100 milliliters
chloroform and washed thrice with water. The organic fractions were
dried over sodium sulfate (10 gram) to remove traces of water. The
solvent was removed on a rotary evaporator at a temperature between
20 and 30 degree Celsius to yield a residue. The residue was
fractionated by column chromatography (15 centimeter length by 4
centimeter diameter) using a dry packed silica gel medium under
laboratory vacuum and fractions were eluted with chloroform.
Fractions were collected based on color and tested for urushiol
presence by a ferric chloride test and GCMS.
Fractions that tested positive for catechol were combined, stripped
of solvent, and resuspended in 100 milliliters hexane and solvent
extraction using 100 milliliters acetonitrile and a continuous
extraction device. Acetonitrile fractions were combined and the
solvent was evaporated therefrom, yielding 0.35 gram of dark green
oil. GCMS analysis showed 92% w/w urushiol. Further purification
was achieved by column chromatography (15 centimeter length by 4
centimeter diameter) using a dry packed thiazole silica gel medium
under laboratory vacuum. The purity was 98%. The concentrated
extract was resuspended in ethanol and stored in a refrigerator (3
degrees Celsius) in order to slow degradation.
EXAMPLE 4
Poison ivy leaves (29 grams) were soaked in 1:1 dichloromethane:
THF (200 milliliters) for approximately 4 hours at room
temperature. The resulting solution was filtered to remove the
leaves, collected in a round bottom flask, and stripped of solvent
using a rotary evaporator. GCMS analysis showed approximately 30%
w/w urushiol. The resulting dark green residue was resuspended in
100 milliliters chloroform and extracted thrice with water using a
continuous extraction device. The organic fractions were dried over
sodium sulfate (10 gram) to remove traces of water. The solvent was
removed using a rotary evaporator at a temperature between 20 and
30 degree Celsius to yield a residue. The residue was fractionated
by column chromatography (15 centimeter length by 4 centimeter
diameter) using a dry packed silica gel medium under laboratory
vacuum and fractions were eluted with chloroform. Fractions were
collected based on color and tested for urushiol presence by a
ferric chloride test and GCMS.
Fractions that tested positive for catechol were combined, stripped
of solvent, and resuspended in 100 milliliters hexane followed by
three rounds of solvent extraction, each round involving extraction
with 100 milliliters of acetonitrile. The acetonitrile aliquots
were combined and acetonitrile was evaporated therefrom, yielding
0.30 gram of dark green oil. GCMS analysis showed 90% w/w urushiol.
The concentrated extract was resuspended in ethanol and stored in a
refrigerator.
EXAMPLE 5
Poison ivy leaves (35 grams) were soaked in THF (200 milliliters)
for approximately 4 hours at room temperature. The resulting
solution was filtered to remove the leaves, collected in a round
bottom flask, and stripped of solvent using a rotary evaporator.
GCMS analysis showed approximately 31% w/w urushiol. The resulting
dark green residue was resuspended in 100 milliliters chloroform
and washed thrice with water. The organic fractions were dried over
sodium sulfate (10 gram) to remove traces of water. The solvent was
removed using a rotary evaporator at a temperature between 20 and
30 degree Celsius to yield a residue. The residue was fractionated
by column chromatography (15 centimeter length by 4 centimeter
diameter) using a dry packed silica gel medium under laboratory
vacuum, and fractions were eluted with chloroform. Fractions were
collected based on color and tested for urushiol presence by a
ferric chloride test and GCMS.
Fractions that tested positive for catechol were combined, stripped
of solvent, and resuspended in 100 milliliters hexane. The
resuspended material was extracted using 100 milliliters of
acetonitrile in a continuous extraction device. Acetonitrile
fractions were combined and acetonitrile was evaporated therefrom,
yielding 0.36 gram of dark green oil. GCMS analysis showed 93% w/w
urushiol. Further purification was achieved by column
chromatography (15 centimeter length by 4 centimeter diameter)
using a dry packed thiazole silica medium, and fractions were
eluted with chloroform under laboratory vacuum. The purity was
97.5%. The concentrated extract was resuspended in ethanol and
stored in a refrigerator.
EXAMPLE 6
Poison ivy leaves (32 grams) were soaked in acetonitrile (200
milliliters) for approximately 4 hours at room temperature. The
resulting solution was filtered to remove the leaves, collected in
a round bottom flask, and stripped of solvent using a rotary
evaporator. GCMS analysis showed approximately 34% w/w urushiol.
The resulting dark green residue was resuspended in 100 milliliters
chloroform and washed thrice with water using a continuous
extraction device. The organic fractions were dried over sodium
sulfate (10 gram) to remove traces of water. The solvent was
removed using a rotary evaporator at temperature between 20 and 30
degree Celsius to yield a residue. The residue was fractionated by
column chromatography (15 centimeter length by 4 centimeter
diameter) using a dry packed silica gel medium with laboratory
vacuum applied, and fractions were eluted with chloroform.
Fractions were collected based on color and tested for urushiol
presence by a ferric chloride test and GCMS.
Fractions that tested positive for catechol were combined, stripped
of solvent, and resuspended in 100 milliliters hexane. The
resuspended material was extracted using 100 milliliters of
acetonitrile in a continuous extraction device. Acetonitrile
fractions were combined and acetonitrile was evaporated therefrom,
yielding 0.31 gram of dark green oil. GCMS analysis showed 93% w/w
urushiol. Further purification was achieved by column
chromatography (15 centimeter length by 4 centimeter diameter)
using a dry packed thiazole silica gel medium, and fractions were
eluted with chloroform under vacuum. The purity was 96.5%. The
concentrated extract was resuspended in ethanol and stored in a
refrigerator
EXAMPLE 7
Poison ivy leaves (60 grams) were soaked in dimethoxyethane (400
milliliters) for approximately 4 hours at room temperature. The
resulting solution was filtered to remove the leaves, collected in
a round bottom flask, and stripped of solvent using a rotary
evaporator. GCMS analysis showed approximately 34% w/w urushiol.
The resulting dark green residue was then resuspended in 100
milliliters ether and washed thrice with water. The organic
fractions were dried over sodium sulfate (20 gram) to remove traces
of water. The solvent was removed using a rotary evaporator at a
temperature between 20 and 30 degrees Celsius to yield a residue.
The residue was purified by column chromatography (25 centimeter
length by 4 centimeter diameter) using a dry packed silica gel
medium with laboratory vacuum applied, and fractions were eluted
with chloroform. Fractions were collected based on color and tested
for urushiol presence by a ferric chloride test and GCMS.
Fractions that tested positive for catechol were combined, stripped
of solvent, and resuspended in 200 milliliters hexane. The
resuspended material was extracted using 100 milliliters of
acetonitrile in a continuous extraction device. Acetonitrile
fractions were combined and acetonitrile was evaporated therefrom,
yielding 0.62 gram of dark green oil. GCMS analysis showed 93% w/w
urushiol. Further purification was achieved by column
chromatography (15 centimeter length by 4 centimeter diameter)
using a dry packed thiazole silica medium, and fractions were
eluted with chloroform under laboratory vacuum. The purity was
98.5%. The concentrated extract was resuspended in ethanol and
stored in a refrigerator.
EXAMPLE 8
Poison ivy leaves (60 grams) were soaked in propanol (400
milliliters) for approximately 4 hours at room temperature. The
resulting solution was filtered to remove the leaves, collected in
a round bottom flask, and stripped of solvent using a rotary
evaporator. GCMS analysis showed approximately 34% w/w urushiol.
The resulting dark green residue was resuspended in 100 milliliters
ether and washed thrice with water. The organic fractions were
dried over sodium sulfate (20 gram) to remove traces of water. The
solvent was removed using a rotary evaporator at a temperature
between 20 and 30 degrees Celsius to yield a residue. The residue
was fractionated by column chromatography (25 centimeter length by
4 centimeter diameter) using a dry packed silica gel medium with
laboratory vacuum applied, and fractions were eluted with
chloroform. Fractions were collected based on color and tested for
urushiol presence by a ferric chloride test and GCMS.
Fractions that tested positive for catechol were combined, stripped
of solvent, and resuspended in 200 milliliters hexane. The
resuspended material was extracted with 100 milliliters of
acetonitrile using a continuous extraction device. Acetonitrile
fractions were combined and acetonitrile was evaporated therefrom,
yielding 0.60 gram of dark green oil. GCMS analysis showed 93% w/w
urushiol. Further purification was achieved by column
chromatography (15 centimeter length by 4 centimeter diameter)
using a dry packed thiazole silica gel medium, and fractions were
eluted with chloroform under laboratory vacuum. The purity was
97.5%. Concentrated extract was resuspended in ethanol and stored
in a refrigerator.
EXAMPLE 9
Poison ivy leaves (35 grams) were soaked in 150 milliliters of
chloroform for approximately 4 hours at room temperature. The
resulting solution was filtered to remove the leaves, collected in
a round bottom flask, and stripped of solvent using a rotary
evaporator. GCMS analysis showed approximately 30% w/w urushiol.
The resulting dark green residue was resuspended in 100 milliliters
chloroform and washed thrice with water using a continuous
extraction device. The organic fractions were dried over sodium
sulfate (10 gram) to remove traces of water. The solvent was
removed using a rotary evaporator at a temperature between 20 and
30 degrees Celsius to yield a residue. The residue was fractionated
by column chromatography (15 centimeter length by 4 centimeter
diameter) using a dry packed silica gel medium under laboratory
vacuum, and fractions were eluted with chloroform. Fractions were
collected based on color and tested for urushiol presence by a
ferric chloride test and GCMS.
Fractions that tested positive for catechol were combined, stripped
of solvent, and resuspended in 200 milliliters hexane. The
resuspended material was extracted with 100 milliliters of
acetonitrile in a continuous extraction device. Acetonitrile
fractions were combined and acetonitrile was evaporated therefrom,
yielding 0.55 gram of dark green oil. GCMS analysis showed 91% w/w
urushiol. Further purification was achieved by column
chromatography (15 centimeter length by 4 centimeter diameter)
using a dry packed thiazole silica gel medium, and fractions eluted
with chloroform under laboratory vacuum. The purity was 94.5%.
Concentrated extract was resuspended in ethanol and stored in a
refrigerator.
EXAMPLE 10
Poison ivy leaves (40 grams) were soaked in 200 milliliters of
pentane for approximately 4 hours at room temperature. The
resulting solution was filtered to remove the leaves, collected in
a round bottom flask, and stripped of solvent on a rotary
evaporator. GCMS analysis showed approximately 42.4% w/w urushiol.
The resulting dark green residue was resuspended in 100 milliliters
chloroform and washed thrice with water using a continuous
extraction process. The organic fractions were dried over sodium
sulfate (10 gram) to remove traces of water. The solvent was
removed using a rotary evaporator at a temperature between 20 and
30 degrees Celsius to afford a residue. The residue was
fractionated by column chromatography (15 centimeter length by 4
centimeter diameter) using a dry packed silica gel medium with
laboratory vacuum applied, and fractions were eluted with
chloroform. Fractions were collected based on color and tested for
urushiol presence by a ferric chloride test and GCMS.
Fractions that tested positive for catechol were combined, stripped
of solvent, and resuspended in 200 milliliters hexane. The
resuspended material was extracted with 100 milliliters
acetonitrile using a continuous extraction device. Acetonitrile
fractions were combined and acetonitrile was evaporated therefrom,
yielding 0.50 gram of dark green oil. GCMS analysis showed 86.5%
w/w urushiol. Further purification was achieved by column
chromatography (15 centimeter length by 4 centimeter diameter)
using a dry packed thiazole silica gel medium, and fractions were
eluted with chloroform under laboratory vacuum. The purity was
91.5%. Concentrated extract was resuspended in ethanol and stored
in a refrigerator.
EXAMPLE 11
Silica (25 grams) was suspended in 200 milliliters of dry toluene
before adding 10 milliliters of 3-aminopropyltriethoxysilane. The
mixture was stirred and refluxed for 24 hours under a nitrogen
atmosphere. The resulting product was filtered and washed twice
with 50 milliliters of toluene, twice with 50 milliliters of
ethanol, and four times with 50 milliliters of dichloromethane. The
product was dried for 6 hours at room temperature under laboratory
vacuum, and then immersed in 200 milliliters of toluene prior to
adding 6.25 milliliters of ethyl-2-bromopropionate. The mixture was
again stirred and refluxed for 24 hours under a nitrogen
atmosphere. The resulting product was filtered and washed twice
with 50 milliliters of toluene, four times with 50 milliliters of
ethanol, and four times with 50 milliliters of dichloromethane. The
resulting product was dried for 6 hours at room temperature under
laboratory vacuum, dispersed in 200 milliliters of dry
acetonitrile, and then 8.3 grams of 2-(thiazol-4-yl)ethanamine was
added. The mixture was stirred and refluxed for 24 hours under a
nitrogen atmosphere. The product was filtered and washed twice with
50 milliliters of acetonitrile, six times with 50 milliliters of
ethanol, and four times with 50 milliliters of dichloromethane.
This final product was dried at room temperature under vacuum to
yield a thiazole silica gel.
EXAMPLE 12
Urushiol congeners were separated as follows. An absorbance
spectrum was obtained using purified extract showing maximum
absorbance at 276 nanometers. High pressure liquid chromatography
analysis was run on a C18 reverse phase column, and individual
urushiol congener peaks were assessed by mass spectrometry (MS).
Dried poison ivy urushiol was dissolved in ethanol and eluted with
a 60 to 100% methanol gradient. Samples were collected and analyzed
by MS. Preparative separation of urushiol components of the
purified poison ivy extract was performed by reverse phase
preparatory FPLC.
EXAMPLE 13
Detoxification of Methylene Chloride Extract--Solvent Removal
Urushiol extract prepared using methylene chloride as the primary
solvent was transferred into a round bottom flask and stripped of
solvent using a rotary evaporator to near dryness. A sample taken
therefrom for GCMS analysis, which indicated the presence of hexane
and methylene chloride in the sample. The extract was thrice
resuspended in approximately 150 milliliters of diethyl ether and
stripped of solvent. Following resuspension in ethanol, the product
contained no detectable amount of hexane or methylene chloride.
EXAMPLE 14
Degradation Study for Clinical Solution Stability
A total of six samples (0.5 milliliter each) of urushiol
preparations made as described herein using methylene chloride as
the primary solvent. Sample concentrations were 50 milligrams per
milliliter, 5 milligrams per milliliter, and 0.5 milligram per
milliliter, and these samples were prepared in duplicate. One
duplicate of each of the three samples was stored at 23 degrees
Celsius and the other was stored at 3 degrees Celsius. Gas
chromatography profiles were obtained periodically over the course
of 249 days to monitor urushiol levels in the samples.
EXAMPLE 15
Gas Chromatography-Mass Spectrometric Analysis of Urushiol
Purified urushiol reconstituted into ethanol was subjected to gas
chromatography followed by the determination of mass charge ratio
on a Agilent Model 5973N GC-MS System with column C8 reverse
column.
EXAMPLE 16
Liquid Chromatographic Analysis of Urushiol
High Pressure Liquid Chromatography (HPLC) analysis was run on C18
reversed phase column, to determine individual urushiol congener
peaks by mass spectrometry on a Agilent Model 1100 LC-MS System.
Dried poison ivy urushiol was dissolved in ethanol and eluted with
a 60 to 100% methanol gradient. Samples were collected and analyzed
by ion-trap mass spectrometry.
The disclosure of every patent, patent application, and publication
cited herein is hereby incorporated herein by reference in its
entirety.
While this subject matter has been disclosed with reference to
specific embodiments, it is apparent that other embodiments and
variations can be devised by others skilled in the art without
departing from the true spirit and scope of the subject matter
described herein. The appended claims include all such embodiments
and equivalent variations.
* * * * *